Thermal Cycle Sequencing Research Articles

X-Ray Investigation on Strength of Thin Films

The behavior of thermal stresses and residual stresses in aluminum and copper thin films were investigated by the diffraction of X-rays and synchrotron radiations. Aluminum films were exposed in an elevated temperature in order to measure the thermal stress behavior in the films. Thermal stresses in the films in thin aluminum and copper films of 10 nm to 1000 nm thickness with and without passivation payer were measured in a sequence of thermal cycles. Stresses in thick films without passivation traced along with a hysteresis loop during thermal cycles, whereas the thin films of nano-meter size with AlN passivation layer behaved in a linear way with the temperature change. Thin copper films with and without AlN passivation layer were used to investigate strengthening mechanism of thin films. Specimens with different thickness of the films were subjected to a cyclic plane bending fatigue test. Residual stresses developed in the films were measured in a sequence of bending cycles to understand the effect of film thickness and passivation layer on mechanical properties of thin films. The film of 500 nm thickness developed less residual stresses in the film than thicker films. A passivation layer was effective to prevent residual stress development in the film.

Annealing effects on leakage current and epilayer doping concentration of p +n junction 4H-SiC diodes after very high neutron irradiation

In this work, we analyzed the annealing effects on silicon carbide (SiC) p +n diodes after very high 1 MeV neutron fluence. The diode structure is based on ion-implanted p + emitter in n-type epilayer with thickness equal to 5 μm and donor doping N D=3×10 15 cm −3. These devices were irradiated with 1 MeV neutrons at four different fluence values, logarithmically distributed in the range 10 14−10 16 cm −2. After irradiation, the epilayer material became more resistive, as indicated by the reduction of the forward and reverse current density at a given voltage. In particular, after irradiation at the highest fluence value, the average leakage current density at 100 V reverse bias decreased from 3 nA/cm 2 to values of the order of 100 pA/cm 2. After a neutron fluence of 1×10 14 cm −2, the epilayer doping concentration decreased to 1.5×10 15 cm −3. The samples underwent a sequence of thermal cycles first at 80 °C and then at 200 °C to verify if a damage recovery occurs in irradiated SiC samples, as in the case of silicon ones. After annealing at 80 °C, the reverse current further decreased, while the depletion voltage remained almost constant. After thermal cycles at 200 °C, the current decreased further and the depletion voltage slightly increased, showing a very low recovery of the damage.

Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing.

Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.

DNA Sequencing by the Dideoxy Method

In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single-stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S-labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high-resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the "labeling/termination" procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional "Sanger" procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers for four-color dideoxy DNA sequencing is also described in detail.

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

FIR Reflectivity, Pyroelectric Effect and Dielectric Constant in 8% Brominated BCCD

Betaine calcium chloride dihydrate is known to exhibit a rich number of commensurate and incommensurate phases (k = δ(T) c*) between a room temperature paraelectric phase (Pnma) and a lower temperature ferroelectric phase (Pn2 1 a). Partial bromination of this compound induces important changes in the phase diagram of the material. This work reports recent results of FIR reflectivity, dielectric dispersion and pyroelectric effect measured on 8% brominated BCCD. In agreement with previous Raman results reported by Schaack and coworkers, the FIR reflectivity (E∥b) spectra (20 cm -1 < ω < 300 cm -1 ) show that the sequence of commensurate and incommensurate phases observed in the pure compound is shifted towards lower temperatures and that the structure remains modulated at lower temperatures (modulation wavenumber δ 1/4 at T = 20 K). The temperature and frequency dependence of the dielectric constants e & and e b , as well as the behaviour of the pyroelectric coefficient λb observed along a sequence of thermal cycles with and without a bias field, indicate a progressive deviation from thermal equilibrium and a subsequent freezing of the structure at low temperatures. The experimental results are discussed within the framework of a phenomenological model based on a Landau-type free energy expansion, previously reported to account for the phase transition sequence observed in the pure crystal under a positive hydrostatic pressure.

Cycle sequencing of filamentous phage DNA using a biotinylated primer and delta Taq DNA polymerase.

We describe a rapid nonradioactive, double-stranded phage DNA sequencing method using ΔTaq DNA polymerase in cycle reaction for phage peptide-display library screening. This procedure is specific, rapid, sensitive and safe for the sequencing of large numbers of phage peptide-display colonies. In addition, thermal cycle sequencing with this chemiluminescent image detection protocol provides an inexpensive method for completing a large amount of sequencing reactions. Consequently, this method is especially useful for those extensively working on peptide mimics using phage display libraries.

A complementary DNA encoding an antigen from Trichinella spiralis muscle larvae and its analog from Trichinella T 5 of bobcat origin: Sequence, cloning and expressions

Reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA encoding an excretory-secretory (ES) antigen with mol. wt 45–50 kDa by SDS-PAGE from T. spiralis muscle larvae. The PCR product was purified by electrophoresis and sequenced by thermal cycle sequencing with primer walking. The cDNA is 890 bp long and encodes a polypeptide of 255 amino acid (AA) residues. Using the same methods, we also recovered a corresponding cDNA from Trichinella T 5, which is 891 bp long and encodes 255 AAs. Comparison of the 2 Trichinella species indicates approximately 2.6% and 2.4% differences between the 2 cDNA sequences and between the 2 deduced AA sequences, respectively.

Thermal cycle dideoxy DNA sequencing.

Thermal cycle dideoxy DNA sequencing eliminates the requirements for independent primer annealing and double-stranded DNA denaturation steps. The method enables sequencing from nanogram amounts of DNA from double-stranded and single-stranded PCR products, and plasmid or phage DNA templates. Thermal cycle sequencing also enables direct sequencing from bacterial colonies or phage plaques. Protocols using the Vent exo- DNA polymerase, helpful suggestions, and a troubleshooting guide are also presented.

Strategies for unambiguous detection of allelic heterozygosity via direct dna sequencing of PCR products: Application to the hla drb1 locus

Background: Many genetic loci exhibit substantial heterogeneity: the human leukocyte antigen (HLA) DRB loci include 139 alleles and the cystic fibrosis transmembrane regulator gene more than 500 known mutations. Identification of alleles at these loci is cumbersome with typical molecular diagnostic methods such as hybridization assays or restriction enzyme analysis. Direct DNA sequencing of polymerase chain reaction (PCR) products is a general approach to complex loci that allows detection of any allele within the nucleotide sequence analyzed. However, direct DNA sequence-based unambiguous identification of heterozygous nucleotide positions using PCR templates is a challenging problem. Methods and Results: The ability of direct DNA sequencing methods to accurately identify HLA DRB alleles was assessed. The authors evaluated the performance of modified T7 and Taq DNA polymerases in isothermal and thermal cycle sequencing of PCR products derived from HLA DRB genes in 235 individuals who were potential donors or recipients of bone marrow transplants. The uniformity of peak intensity and ability to identify heterozygous nucleotide positions was similar when either AmpliTaq FS- or Sequenase DNA polymerase-derived electropherograms were prepared. The modified Taq DNA polymerase allowed the use of unpurified, double-stranded PCR templates. Furthermore, this enzyme could be used in less laborious, less costly cycle sequencing assays coupled with automated fluorescent detection methodology. Direct sequencing performed with either enzyme allowed unambiguous identification of DRB1 alleles, resolution of difficult heterozygous combinations, and recognition of new alleles. Conclusions: The direct DNA sequencing methods employed here for HLA allele identification are relatively efficient and semiautomated, and may be reasonably considered as a general approach to other complex molecular diagnostic problems, especially when coupled to simplified sequencing chemistries allowing cycle sequencing. (Mol Diagn 1996 Jun;1(2):89-98)

The two-way memory effect by the pre-strain training method in a 50Ti40Ni10Cu (at.%) alloy

The two-way memory effect (TWME) can be settled after a training of the shape memory alloy apt to stabilize the preferential growth (shrinkage) of martensite domains by decreasing (increasing) temperature. That appears as a consequence of a pattern of line defects which act as nucleation sites of selected variants growth. Though several training procedures have been suggested, a clear cut view of the most efficient one has not yet been reached. Recently a pre-strain method has been adopted to settle the TWME in TiNi alloys, with the highest reversible strain obtained for a plastic pre-strain introduced at temperatures (T) M{sub s}TA{sub f}. The TWME on 50Ti40Ni10Cu (at%) alloy has already been investigated with a training procedure based on a sequence of thermal cycles under applied stress involving the B2(parent) {leftrightarrow} B19(orthorhombic) transformation. Interest is here given to the pre-strain training TWME in a 50Ti40Ni10Cu (at%) alloy introducing the plastic prestrain either in monoclinic (B19{prime}), or in orthorhombic (B19), or in parent (B2) phase in the range of stress induced martensite.

Distribution of the DNA adducts of 2‐amino‐3–methylimidazo[4,5–f]quinoline and 2–amino‐1–methyl‐6–phenylimidazo[4,5–b]pyridine in the supF gene as determined by polymerase arrest assay

The distribution of the adducts of the cooked meat-derived heterocyclic amines 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) was examined in the supF gene of PSP189 by a polymerase-arrest assay using thermal-cycle sequencing. The reactive N-acetoxy metabolites of both compounds showed an overwhelming preference for reacting with guanine residues in the supF gene of the shuttle vector pSP189. The distribution of the IQ and PhlP guanine adducts was not random; instead, patterns of adduct hot-spots and cold-spots were observed. There was a striking similarity between both compounds in their preferred sites of adduct formation. The finding that IQ and PhlP adducted to guanine concurred with previous results showing that the target sites for IQ and PhlP mutations in supF were also at guanine. However, the adduct hot-spot sites were not predictive of the known sites of mutation hot-spots. In addition, despite the similarity in adduct hot-spots for IQ and PhlP, their reported mutation spectra in the supF gene were different. Factors in addition to adduct location therefore appear to play a role in the mutation spectra induced by the heterocyclic amines in the supF gene.

Integration of 28 STSs into the physical map of human chromosome 18.

Genes on human chromosome 18 are associated with familial glucocorticoid deficiency (MC2R), pemphigus vulgaris (DSG3) and foliaceus (DSG1), familial amyloidosis (TTR), colorectal carcinoma (DCC), erythropoietic protoporphyria (FECH), follicular lymphoma (BCL2, FVT1), and congenital methemoglobinemia (CYB5). As the resolution of human genetic maps improves, linkage between other diseases and specific regions of chromosome 18 will occur. A physical map of human chromosome 18 will prove useful in identifying candidate genes that are associated with these disorders. Using various physical and genetic mapping techniques, over 35 genes and 19 expressed sequence tags (ESTs) are assigned to human chromosome 18. Most of these genes and several of the ESTs were sublocalized using a well-defined panel of somatic cell hybrids that contain different segments of human chromosome 18. Despite recent efforts, progress in mapping human chromosome 18 has lagged behind that achieved for other chromosomes. Thus, the purpose of this study was to integrate 9 new transcriptional tags [8 brain ESTs (8) and the melanocortin 4 receptor (MC4R) (3)] and 19 simple sequence repeats (SSRs) into the physical map of human chromosome 18. The SSRs were isolated by screening genomic DNA libraries constructed in M13mp18 vectors with oligonucleotide probes that detected dinucleotide d(CA)- andmore » tetranucleotide-repeat motifs. DNA sequences of clones that contained microsatellite repeats were obtained by thermal-cycle sequencing, and STSs were developed from clones that contained numerous repeats. STSs that identified highly polymorphic loci in eight unrelated CEPH parents were used for genotyping. Results of linkage analyses and estimates of heterozygosity for these markers will be reported. 9 refs., 1 fig., 1 tab.« less

Comparison of nucleotide sequences among hantaviruses belonging to the same serotype: an analysis of amplified DNA by thermal cycle sequencing

The hantavirus genus, belonging to the bunyaviridae family, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. Previously, we reported the use of the polymerase chain reaction (PCR) for grouping hantavirus isolates by using four sets of primers specific to each serotype. Our PCR typing results agreed with those of serological typing. The present study makes use of thermal cycle sequencing to sequence PCR-amplified DNA products in order to determine the level of similarity among members of the same serotype. We show that members of Hantaan and Seoul serotypes are over 92% homologous, irrespective of their host and geographical origin. Puumala sequences show a degree of homology ranging from 80 to 98%. Despite the variation in sequence at the nucleotide level, amino acids show an even higher level of conservation.

Preparation of Templates for DNA Sequencing

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a lambda-derived phage as a source of DNA, a protocol for preparing lambda phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double-stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.